Process and media for the growth of human epithelia

ABSTRACT

Novel media and methods are disclosed for the in vitro formation of a histologically complete human epithelium. The media are serum-free, companion cell or feeder layer free and organotypic, matrix free solutions for the isolation and cultivation of clonally competent basal epithelial cells. The media and methods of the invention are useful in the production of epithelial tissues such as epidermis, cornea, gingiva and ureter.

ADVANCEMENT OF EXAMINATION

Applicants respectfully request that this application be made specialunder 37 C.F.R. §1.102(d). This request is accompanied by the petitionfee set forth in 37 C.F.R. §1.17(i).

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/500,744,filed Jul. 11, 1995, now U.S. Pat. No. 5,686,307 entitled: SERUM-FREEMEDIUM FOR USE IN THE FORMATION OF A HISTOLOGICALLY COMPLETE LIVINGHUMAN SKIN SUBSTITUTE; which is a continuation of U.S. Ser. No.08/318,221, filed Oct. 5, 1994, now abandoned; which is a continuationof U.S. Ser. No. 08/184,905, filed Jan. 21, 1994, now abandoned; whichis a continuation of Ser. No. 08/063,247, filed May 18, 1993, nowabandoned; which is a divisional of U.S. Ser. No. 07/471,976, filed Jan.29, 1990, now U.S. Pat. No. 5,292,655. U.S. Pat. Nos. 5,292,655 and5,686,307 are both incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The field of the invention is in biology and more specifically in thesubspecialty of cell biology. The invention relates to a process andcell culture media for the growth of human epithelia, such as gingivalepithelium, ureteral epithelium and corneal epithelium.

BACKGROUND OF THE INVENTION

The epithelium is the membranous cellular tissue that covers the surfaceor lines a tube or cavity of an animal body. The epithelium serves toenclose and protect the other parts of the body and may producesecretions and excretions and may be associated with assimilation asseen in the gastrointestinal tract. The epithelium is one of the fourprimary tissues of the body, which constitutes the epidermis and thelining of respiratory, digestive and genitourinary passages. The cornea,which is the transparent part of the coat of the eyeball that covers theiris and pupil and admits light to the interior, is also a tissue thatis made of epithelial cells.

The functions of epithelia are varied and include: (1) protectivefunction, by completely covering the external surface (including thegastrointestinal surface, the surface of the whole pulmonary treeincluding the alveoli and the eye); (2) secretory function, by secretingfluids and chemical substances necessary for digestion, lubrication,protection, excretion of waste products, reproduction and the regulationof metabolic processes of the body; (3) absorptive function, byabsorbing nutritive substances and preserving water and salts of thebody; (4) sensory function, by constituting important parts of senseorgans, especially of smell and taste; and (5) lubricating function, bylining all of the internal cavities of the body, including theperitoneum, pleura, pericardium and the tuncia vaginalis of the testis.

The growth of human epithelial cells without the use of companion-cells,feeder layers, serum components or organotypic substrates is theadvancement in the state of the art that is this invention.Traditionally, tissue culture of normal epithelial cells has beenattempted in a variety of commercially available media designed for thegrowth of less fastidious types of cells, i.e., malignant cellstransformed in vitro from cell lines derived from human or non-humantissues, cell lines developed from human or non-human tumors, or celllines developed for human or non-human embryonic mesenchymal cell types.In contrast, the culture of normal human epithelial stem cells haspresented many difficulties not the least of which is the inexorabletendency for these cells to undergo uncontrolled, irreversible, terminaldifferentiation with the consequent loss of cell division capacity.

A significant development made by Tsao et al. is the formulation of anutrient medium supplemented with specified growth factors and hormonesallowed for the growth of human epidermal cells. See Tsao, M. C. et al.,J Cellular Physiol. 110:219-229 (1982). The Tsao medium has beendesignated MCDB 152. Further refinements of this medium lead to thedevelopment of a medium known as MCDB 153. See Boyce, S. T. and Ham, R.G., J Invest. Dermatol. 81:33-40 (1983). The use of these mediapermitted a more accurate characterization of the necessary growthfactors, hormones and Ca²⁺ requirements for retention of high cloningefficiency which is necessary to maintain proper genetic programming forcontinued subculture of pluripotent basal epidermal stem cells. SeeWille, J. J. et al., J Cellular Physiol. 121:31-44 (1984).

The use of serum in cell culture medium provides a complex mixture ofgrowth factors and differentiation-inducing factors. See Pittelkow, M.R. et al., J Invest, Dermatol. 86:410-417 (1986). Pittelkow et al.reported that serum, known to contain fibroblastic cell growth factors,e.g., platelet-derived growth factor, was an inhibitor of basalepidermal cell growth. Further, the differentiation-inducing factors inserum could be equated with serum's content of β-transforming growthfactor, (β-TGF). See Shipley, S. D. et al., Cancer Res. 46:2068-2071(1986). It has also been reported that normal human keratinocytesactually produce their own growth factors. That is, proliferating basalcells are stimulated to secrete α-transforming growth factor (α-TGF) inthe presence of added epidermal growth factor (EGF) and decreaseproduction of α-TGF at high cell densities near confluence. Under thelatter condition, the arrested cells secrete an inactive form of β-TGF.See Coffey, R. J. et al., Nature 328:817-820 (1987). Theseconsiderations led the inventor to the idea that the natural mechanismof growth stimulation and its regulation in cultured epithelia cellscould be accomplished through manipulation of the various mediacomponents and that such manipulation would also eliminate the need foran organic substrate or organotypic matrix as well.

Judd et al. discuss a keratinocyte growth medium designatedkeratinocytes-SFM in an article entitled: "Culture of HumanKeratinocytes in Defined Serum Free Medium", Focus, 19, No. 1, Pgs. 1-5.This serum-free media is also disclosed in a Gibco Product brochure.However, the actual composition of the SFM media is not disclosed otherthan it does not contain the growth promoting additives insulin,epidermal growth factor and fibroblast growth factor.

An article by Wille et al., in J Dental Research, 68:1019 (1989)entitled "Serum Free Cultures of Normal Human Gingival Keratinocytes(HGK)" discusses the successful in vitro culturing of human gingivalkeratinocytes in MCDB 153 medium, supplemented with 0.1 mM ethanolamine,0.1 mM phosphoethanolamine, 0.5 μm hydrocortisone, 5 ng per ml epidermalgrowth factor, 5 μg per ml insulin and 35 μg per ml bovine pituitaryextract protein where the presence of these proteins is necessary, buttheir function is unknown in this heterogeneous tissue extract mixture.Wille et al. in The Journal of Cellular Physiology, 150:52-58 (1992) inan article entitled "Effects of Growth Factors, Hormones, BacterialLipopolysaccharides and Lipotechoic Acids on the Clonal Growth ofUrethreal Epithelial Cells in Serum Free Culture", discloses the use ofF-12 media containing bovine pituitary extract and bovine serum albuminfor culturing cells isolated from human ureters, again where such tissueproducts have necessary but unknown effective components.

Chopra et al. in the Journal of Cellular Physiology, 130:173-181 (1987)entitled: "Propagation of Differentiating Normal Human TracheobronchialEpithelial in Serum Free Medium" discloses the use of a medium similarto MCDB 151 except that it contains 5.4 mg per ml HEPES, 6.1 mg per mlsodium chloride, 0.3 mg per ml sodium acetate and 1 mg per ml sodiumbicarbonate. These changes lowered the final osmolarity of the disclosedmedium to 290 mosmols. The concentration of HEPES in the Chopra et al.solution was 28 mM.

U.S. Pat. No. 5,328,844 to Moore discloses a culture medium useful forestablishing, growing and maintaining mammalian cells in culture, inparticular for the establishment of culture of human, normal andmalignant cells. The claimed media contains 4,500 mg per liter of HEPESand 5 mg per liter of insulin. This patent does not relate to nordisclose media useful for growth of normal epithelial cells.

In an article by Boisseau et al. entitled "Production of EpidermalSheets in a Serum Free Culture Medium: A Further Appraisal of the Roleof Extracellular Calcium", Journal of Dermatological Science, 3 (1992),111-120, the author discloses the serum-free media (MCDB 153) to growkeratinocyte monolayers in clonogenic conditions. The effect ofextracellular calcium and temperature on proliferation anddifferentiation of cultured keratinocytes was investigated.

U.S. Pat. No. 4,673,649 to Boyce et al. discloses a basal medium whichwas MCDB 152 supplemented with epidermal growth factor, transferin,insulin, hydrocortisone, ethanolamine, phosphoethanolamine andprogesterone to obtain a medium for growth of human keratinocytes. Theinventor of the present application in U.S. Pat. No. 5,292,655demonstrates that progesterone inhibits optimal growth.

Wilke et al. in "Biologic Mechanisms for the Regulation of Normal HumanKeratinocyte Proliferation and Differentiation", American Journal ofPathology, Vol. 131, No. 1, April, 1988, describe a serum-free mediumwith low calcium concentrations on the order of 0.1 mM. These studies ofWilke et al. actually used MCDB 153 medium supplemented with insulin,EGF and protein of bovine pituitary extract where any effectivecomponents are unknown in the extract.

U.S. Pat. No. 5,232,848 to Wolfe et al. discloses a nutrient medium forboth high and low density culture of a wide variety of non-epithelialcell lines and cell types. This patent discloses and claims azwitterionic buffer such as HEPES at a concentration of 2.5×10⁻² moles.

Boyce et al. in U.S. Pat. No. 4,940,666 discloses and claims a growthmedium which is free of transferin, comprising complete MCDB 153, EGFand insulin.

Nissley et al. in "Growth and Differentiation of Cells in a DefinedEnvironment", pgs. 337-344 discloses that cells of embryonic and fetalorigin produce IGF-1 and IGF-2 which may be important for the control ofembryonic and fetal growth. The authors also suggest that the use ofthese cells could potentially stimulate the growth of the same orneighboring cells and thereby avoid the inclusion of such growth factorsin a culture medium.

Boyce et al. in "Calcium Regulated Differentiation of Normal HumanEpidermal Keratinocytes in Chemically Defined Clonal Culture and SerumFree Serial Culture", in The Journal of Investigative Dermatology,81:33S-40S (1983) discloses MCDB 153 supplemented with a number ofgrowth factors and an optimum level of calcium at 0.3 mM for colonyforming efficiency and a high calcium concentration of 1.0 mM forinduction of stratification and terminal differentiation.

U.S. Pat. No. 5,326,699 to Torishima et al. discloses a serum-freemedium for culturing animal epithelial cells comprising 8-14 mg per ml(53.6 mM -93.8 mM) of methionine, up to 0.1 mM of calcium in the form ofcalcium chloride and other conventional ingredients such as glucose,growth factors, buffers and the like.

Pellegrini in "Long Term Restoration of Damaged Corneal Surfaces withAutologous Cultured Corneal Epithelium", Lancet, Vol. 349 (1997)discloses the culturing of corneal cells in Dulbecco, Vogt, Eagle's andHam's F-12 Media containing fetal bovine serum, insulin, transferin, EGFand cholera toxin. The authors' report that cells isolated from thecentral cornea (limbus) and bulbar conjunctiva could be grown in vitroand then transplanted to the human host.

U.S. Pat. No. 4,304, 866 to Green et al. discloses an in vitro methodfor the formation of epithelial sheets from cultured keratinocytes. TheGreen method uses a serum containing medium and a feeder layer of murine(mouse) fibroblast cells to accomplish cell growth and differentiation.This procedure has serious limitations for large scale production ofhuman epithelium as the use of serum inextricably confounds the cultureof purely basal cells with the dynamics of serum-induceddifferentiation. The net result is that subcultivation of such culturesyields low (<5%) clonal efficiencies preventing step wise large scalebuild up of uncommitted pluripotent basal cells as a prelude to theirconversion into usable sheets of transplantable,histologically-complete, human epithelium. Moreover, the process ofGreen et al. does not describe the formation of a histologicallycomplete epidermis. The Green et al. procedure forms an epidermislacking a stratum corneum which is necessary for maximizing the utilityof the tissue.

Prior art methods have achieved a complete epidermis, but only in thepresence of a complete skin starter sample and serum-containing mediathat are combined with an organotypic substratum containing growthfactors produced by companion cells as disclosed in U.S. Pat. No.4,485,096. The use of any organotypic substrate as well as feeder orcompanion cell types, e.g. fibroblasts, seriously limits the resultingproducts safety and economic viability. See Nanchahal, J. et al. inLancet II(8656):191-193, (1989).

In order to remedy these deficiencies, the inventor has dispensed withserum-containing media, eliminated any substratum support, dispensedwith the requirement for innumerable skin starter samples, and designeda novel and unobvious medium capable of supporting the growth anddevelopment of a complete epithelium. Moreover, the identification ofessential process steps leading to a functional epithelium has beendiscovered and can be monitored with specific monoclonal antibodies. Theprior art media which contain undefined serum and/or feeder cell factorsand/or organotypic substrates and millimolar concentrations of Ca²⁺,high levels of buffers, inadequate levels of amino acids and incorrectosmolalities were not designed for the unlimited proliferation ofundifferentiated basal cells. The prior art media allows cultures tospontaneously undergo maturation and uncontrolled differentiation. Incontrast, the serum-free media described in this invention produces acomplete epithelium.

SUMMARY OF THE INVENTION

There is disclosed an aqueous solution for isolating epithelial cellsfrom animal tissue, said solution comprising:

a) glucose at a concentration of about 10 mM;

b) N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) (HEPES) at aconcentration of 16-22 mM;

c) sodium chloride at a concentration of 90-140 mM;

d) potassium chloride at a concentration of about 3 mM;

e) sodium orthophosphate (Na₂ HPO₄.7H₂ O) at a concentration of 1 mM;

f) phenol red at a concentration of 0.0033 mM;

g) about 100 units of penicillin per ml of solution;

h) about 100 units of streptomycin per ml of solution; and

i) one component selected from the group consisting of:

(i) trypsin at a concentration of 0.1%-0.2% w/v; and

(ii) soy bean trypsin inhibitor at a concentration of 0.1-1.0% w/v.

The aqueous solution for the isolation of the basal epithelial cells orcell competency solution (CCS) is actually two solutions, the firstbeing a solution containing trypsin to digest the cells of interest fromother cellular tissue and the second being a solution containing asoybean trypsin inhibitor to stop the digestion of the tissue.

In a more preferred embodiment of the cell competency solution, thesodium chloride is at a concentration of 100 to 130 mM; the HEPES is ata concentration of 18 to 21 mM; the trypsin is at a concentration of0.12 to 0. 18% w/v in the digestion solution and the soybean trypsininhibitor is at a concentration of 0.3 to 0.8% w/v in the second CCS.

The present invention also relates to a method for the isolation ofbasal epithelial cells from animal tissues, said method comprising thesteps of:

a) obtaining animal epithelium;

b) comminuting said epithelium;

c) placing said comminuted epithelium in the cell competency solutiondescribed above containing trypsin at a temperature and for a timesufficient to allow separation of the basal epithelial cells from theepithelium;

d) collecting said epithelial cells; and

e) passaging said basal epithelial cells to the CCS containing soybeantrypsin inhibitor.

It should be understood that the method described above uses two (2) CCSsolutions:

(1) a solution containing trypsin to digest the tissue; and (2) asolution containing a soy bean trypsin inhibitor to terminate thedigestion of the tissue.

In its broadest sense, the present invention relates to the use of aserum-free medium for culturing animal epithelial cells comprising:

a) N-(2-OH-ethyl-)piperazine-N'-(2-ethane-sulfonic acid) at aconcentration of 14-22 mM;

b) sodium chloride at a concentration of 100-120 mM;

c) histidine at a concentration of 0.1-0.25 mM;

d) isoleucine at a concentration of 0.05-0.5 mM;

e) methionine at a concentration of 0.1-0.5 mM;

f) phenylalanine at a concentration of 0.1-0.5 mM;

g) tryptophan at a concentration of 0.05-0.5 mM; and

h) tyrosine at a concentration of 0.1-0.5 mM.

This serum-free medium of the invention is sometimes hereinafterreferred to as the BASAL medium.

There is further disclosed a serum-free medium for culturing animalepithelial cells comprising:

a) N-(2-OH-ethyl-)piperazine-N -(2-ethane-sulfonic acid) at aconcentration of 14-22 mM;

b) sodium chloride at a concentration of 100-120 mM;

c) histidine at a concentration of 0.1-0.25 mM;

d) isoleucine at a concentration of 0.05-0.5 mM;

e) methionine at a concentration of 0.1-0.5 mM;

f) phenylalanine at a concentration of 0.1-0.5 mM;

g) tryptophan at a concentration of 0.05-0.5 mM;

h) tyrosine at a concentration of 0.1-0.5 mM; and

i) insulin like growth factor -1 at a concentration of 0.3-30 ng/ml.

The serum-free medium described above is sometimes referred to asHECK-109 FS.

In addition, there is disclosed a serum-free medium for culturing animalepithelial cells comprising:

a) N-(2-OH-ethyl-)piperazine-N'-(2-ethane-sulfonic acid) at aconcentration of 14-22 mM;

b) sodium chloride at a concentration of 100-120 mM;

c) calcium²⁺ ions at a concentration of 0.7-3.0 mM;

d) histidine at a concentration of 0.1-0.25 mM;

e) isoleucine at a concentration of 0.05-0.5 mM;

f) methionine at a concentration of 0.1-0.5 mM;

g) phenylalanine at a concentration of 0.1-0.5 mM;

h) tryptophan at a concentration of 0.05-0.5 mM;

i) tyrosine at a concentration of 0.1-0.5 mM; and

j) beta-transforming growth factor at a concentration of 3.0-30 ng/ml.

This inventive serum-free medium is sometimes hereinafter referred to asHECK- 109 DM.

There is further disclosed a serum-free medium for culturing animalepithelial cells comprising:

a) N-(2-OH-ethyl-)piperazine-N'-(2-ethane-sulfonic acid) at aconcentration of 14-22 mM;

b) sodium chloride at a concentration of 100-120 mM;

c) calcium²⁺ ions at a concentration of 0.7-3.0 mM;

d) histidine at a concentration of 0.1-0.25 mM;

e) isoleucine at a concentration of 0.05-0.5 mM;

f) methionine at a concentration of 0.1-0.5 mM;

g) phenylalanine at a concentration of 0.1-0.5 mM;

h) tryptophan at a concentration of 0.05-0.5 mM;

i) tyrosine at a concentration of 0.1-0.5 mM; and

j) linoleic acid at a concentration of 1-15 μg/ml.

The above described serum-free medium according to the invention issometimes hereinafter referred to as HECK-109 CM.

The present invention also relates to a method for the formation of ahistologically complete, stratified animal epithelium using the mediadescribed herein. More specifically, there is disclosed a method for theformation of a histologically complete, stratified human epitheliumcomprising the steps of:

a) isolation of basal stem cells from animal epithelium using the CCSthat contains trypsin;

b) recovering said basal stem cells using CCS that contains soy beantrypsin inhibitor;

c) culturing said isolated basal stem cells in HECK-109 FS medium toform a confluent sheet of undifferentiated epithelial tissue;

d) culturing said sheet of undifferentiated epithelial tissue inHECK-109 DM to form a sheet of differentiated and stratified tissue; and

e) culturing said differentiated and stratified tissue in HECK-109 CM toform a cornified epithelium.

In a preferred embodiment, the method of the present invention forms ahistologically complete human skin.

The invention further relates to the formation of a differentiated andstratified tissue wherein the method is set forth above with theomission of step e) wherein the cornified epithelium layer is formed.The inventive method wherein cornification is omitted is preferablyapplied to tissue such as cornea and gingiva.

DETAILED DESCRIPTION OF THE INVENTION

The epithelial cells that can be advantageously cultured with the mediaof this invention include adult epidermal keratinocytes, adult cornealepithelial cells, ureteral epithelial cells, gingival keratinocytes,fetal epithelial cells and the like. As discussed in the Backgroundsection, the cell culture media according to the invention arebeneficial in growing any epithelium such as the cornea of the eye andlinings of the respiratory, digestive and genitourinary tissues.

The present invention also relates to a method of culturing animalepithelia comprising culturing said cells in the serum-free mediadisclosed above.

The important aspects of the inventive media include the low levels ofHEPES, modified levels of amino acids and a particular range ofosmolalities. The acceptable osmolarities of the media of this inventioncan range from 275 to 310 milliosmols per liter of solution (mosmols).Osmolarity is the concentration of an osmotic solution when measured inosmols or milliosmols per liter of solution. The inventor has also foundthat the reduced levels of HEPES, the sodium chloride concentration,(which is directly related to osmolarity) and the concentration of thesix (6) amino acids, allows for the omission of serum and any foreignprotein factor in the medium and is the basis for the basal mediumhereinafter designated HECK-109. Further, an additional point of noveltyresides in the use of insulin like -1 growth factor at a concentrationof 0.3-30 ng/ml for the fully supplemented growth medium hereinafterdesigned HECK-109 FS. Additional points of novelty relate to thecalcium²⁺ ion concentration of 0.7-3.0 mM and the inclusion ofβ-transforming growth factor at a concentration of 3.0-30 ng/ml for thedifferentiation medium hereinafter designated HECK-109 DM. An additionalinventive media comprises linoleic acid at a concentration of 1-15 μgper ml for cornification of the reformed tissue hereinafter designatedHECK-109 CM.

Admittedly, the prior art is replete with numerous cell culture media.For example, the previously discussed Wolfe, Boyce, Wilke and MCDB mediaare well known and commercially available. However, none of thesereferences have suggested or disclosed the improvements the inventor hasdiscovered herein. Those improvements relate to the reduced level ofHEPES in combination with the specific and specified levels of six verycritical amino acids. Further, while many of the references recite thatthey are serum-free, they are in fact not tissue-extract free as thevarious prior art media are taught to utilize various tissue extractssuch as bovine pituitary extract.

Most of the major nutrients and other factors essential for cell growthare known and have been used previously and in many permutations.However, the concentrations of certain specific components have beennewly formulated for the media of this invention. The components havenot merely been optimized but rather a significant discovery has beenmade in that the components of HEPES and the amino acids are aninterrelated set of factors and enhancers for the growth of humanepithelial cells. The inventor herein has also discovered that thisinterrelationship of the various components can also avoid the use offeeder layers such as mouse cells, which are used to produce serum-likegrowth factors. The novel media of the invention also allows for theavoidance of bovine pituitary extract as taught and suggested by theprior art. As will be demonstrated below, these changes to the mediahave a profound effect on the media's ability to allow for prolific cellgrowth and the ultimate differentiation of the cells into a completeepithelium.

An additional aspect of the present invention resides in the discoverythat the prior art levels of the aromatic amino acids (histidine,phenylalanine, tryptophan and tyrosine) presented the cultured cellswith a rate limiting amount of these vital nutrients. As such, thepresent inventor has included in his media significantly differentamounts of each of these amino acids. Also critically important is theratio of the amino acids to each other as the ratios impart the abilityof this medium to allow the cultured cells to thrive and form aconfluent sheet of tissue without the need for serum components orcomponents derived from serum.

Thus, there is disclosed the formulation and use of novel mediums whichhave been differently supplemented to provide for the achievement ofcellular differentiation of pluripotent basal epithelium cells to afully differentiated human epithelium in vitro. The various media havebeen designated: (i) CCS for cell competency solution; (ii) HECK-109,the basal medium for cell starting; (iii) HECK-109 FS(fully-supplemented) medium for control over cellular growth; (iv)HECK-109 DM (differentiation medium) for the induction ofdifferentiation; and (v) HECK-109 CM (cornification medium) designed forthe induction of cellular differentiation to form a cornification layer.The invention also relates to a sequential process for the in vitroconstruction of a histologically-complete living epithelium in a totallyserum-free medium. The process and media of the invention does notrequire feeder layer cells and is also matrix-free (i.e., collagen orother organotypic matrix).

The nutrient basal medium designated HECK-109 has as criticalcomponents: (i) N-(2-OH-ethyl-)piperazine-N'-(2-ethane sulfonic acid)(hereinafter "HEPES") at 14-22 mM; ii) NaCl at 100-120 mM; and (iii) six(6) key amino acids at about the following concentrations: histidine at1.0-2.5×10⁻⁴ M; isoleucine at 0.5-5.0×10⁻⁴ M; methionine at 1.0-5.0×10⁻⁴M; phenylalanine at 1.0-5.0×10⁻⁴ M; tryptophan at 0.5-5.0×10⁻⁴ M; andtyrosine at 10-5.0×10⁻⁴ M. Taken together, HEPES, NaCl and the six (6)key amino acids are superior to any previous media or similar design, intoxicity, osmolarity and support of clonal growth of basal epithelialcells. All media of this invention, except for the CCS, have anosmolarity of between 275 to 310 mosmols.

The second media is a growth medium for undifferentiated basalepithelial cells and is based on HECK-109 basal medium and hereindesignated HECK-109 FS. HECK-109 FS consists of HECK-109 supplementedwith insulin-like growth factor-1 (IGF-1) at 0.3-30 ng/ml. This mediumis selective for the growth of normal human epithelial cells and isessential for Phase I of the culture growth in that it supports theformation of a hole-free monolayer (intack sheet) of undifferentiatedepithelial cells while suppressing growth-arrest and any significantdecline in clonogenic potential. These properties are unlike anyprevious media used to support proliferation for basal epithelial cells.

The third medium is HECK-109 DM and is a differentiating growth mediumbased on HECK-109 FS with the addition of beta-transforming growthfactor at a concentration of 3.0 to 30 ng/ml. As detailed in Example 4,the induction of synchronous growth arrest, commitment to terminalepithelial differentiation and formation of a supra-basal cell layersuperimposed on top of a proliferation-competent basal cell layer isachieved by replacement of HECK-109 FS with HECK-109 DM. HECK-109 DM isalso HECK-109 FS with Ca²⁺ at a concentration of about 0.7 to 3.0 mM.Addition of β-TGF is required to arrest basal epithelial cells through apathway that prepares the monolayer for induction of stratification, astep under the joint control of EGF (1-5 ng/ml), β-TGF (3-30 ng/ml) andCa²⁺ (0.6-3.0 mM). HECK-109 DM is replaced by HECK-109 CM(cornification-inducing medium) when a cornified layer is required inthe epithelium. HECK-109 CM is based on HECK-109 FS minus the proteinfactors and with the addition of linoleic acid at 1-15 μg/ml and Ca²⁺ions at 0.7-3.0 mM.

The present invention also relates to a method using CCS whereinclonally competent basal epithelial cells are isolated from humanepithelium by the procedures set forth in Example 1. The inventivemethod and CCS allows for the recovery of a unique subpopulation ofbasal cells which differs from basal cells obtained using othertechniques and media. Separation of these unique basal cells from thestarting epithelium is accomplished by digestion in CCS containing0.1-0.2% trypsin (w/v). CCS is designed to permit the initial isolationof a subpopulation of clonally competent basal epithelial cells thatretain a high level clonality. This is due to the low toxicity of theCCS with improved osmolarity. CCS according to this invention differsfrom all other isolation solutions for such cells. The approximatecomposition of CCS is as follows: glucose at about 10 mM; KCl at about 3mM; NaCl at 100-120 mM; Na₂ HPO₄.7H₂ O at about 1 mM; phenol red atabout 0.0033 mM; HEPES at 16-22 mM; about 100 units both of penicillinand streptomycin and SOTI at 0.2-2.0% (w/v) or trypsin at 0.1-0.2% w/v.It should be noted that CCS containing trypsin is used to digest theepithelium and the released basal cells are harvested in the CCScontaining SOTI. The isolated competent basal cells are then seeded intoHECK-109 FS at about 1-5×10 ³ cells/cm² and are amplified to a densityof 2×10⁴ to 1×10⁵ cells/cm² serial passaging.

For large scale preparation of tissue, the undifferentiated basal cellsare amplified by serial propogation in HECK-109 FS until a sufficientquantity of tissue has been obtained. This is known as Phase I. In thenext step, the cells are differentiated by replacement of HECK-109 FSwith HECK-109 DM, at this time the culture has attained a confluent celldensity (1-2×10⁵ cells/cm²). This step is known as Phase II. The cellsare cultured under HECK-109 DM for about 1 week, during which time thecells complete their commitment to differentiation, stratification andkeratinization.

Finally, in those cases where a cornified layer is required in theepithelium, the HECK-109 DM is replaced by HECK-109 CM. This is known asPhase III.

The present invention also discloses a process wherein a viable, andcompletely reformed human epithelium can be grown in culture byfollowing a sequence of steps heretofore unknown using cell culturemedia that do not contain serum, feeder cells or organotypic substrates.The starting material (tissue) can be obtained from fetal tissue,neonatal foreskins and adult epithelial and requires only about 0.1 to 2cm². More preferably, the starting material should be at least 1-2 cm².Representative of other epithelial cells that can be used in thisinvention are those tissues obtained from cornea, ureter and gingiva. Infact, any epithelium of an animal body can be used to obtain the basalcells which are then cultivated and differentiated using the process andmedia of this invention.

Phase I typically amplifies the initial input of basal cells by a factorof 100,000. Typically, this requires less than two weeks and providesenough basal cells to eventually form about 2 to 3 square meters ofviable cultured epithelium.

The reformed epithelium produced in accordance with the inventionconsists of at least two cellular layers. Representative is theepidermis which consists of three histologically recognizable andantibody-identifiable cell layers, a bottom-most cell layer (stratumgerminativum), a spinous cell layer (stratum spinosum) above it, and atop-most granular cell layer (stratum granulosum), but no formation of acornified stratus (stratum corneum), or bordering layers, (e.g., statumlucidum and stratum disjunction). This development requires a thirdphase of culture. The second phase culture, characterized by anincomplete epidermis, will persist in culture for an extended period(>30 days). It will, however, lose the capacity to convert to a completeepidermis which is prevented by replacement of HECK-109 DM with HECK-109CM.

The present invention produces a completely differentiated humanepithelium in a manner that is vastly superior to any prior art methodemploying serum-containing media and/or feeder layer support and/ororganotypic matrices. The present invention also affords the possibilityof intervening at any of the crucial steps in the process that allowsone to augment the cellular content of one of the living versusnonliving cell layers. Finally, the ease of amplifying the initial inputthrough rapid serial cell culture makes this the choice method forinstituting autografts for severe burn patients. The present inventionalso provides a long term solution to patients in need of a cornealtransplant, gingiva repair, ureter reconstruction and the like.

The following Examples are intended to be illustrative and notlimitative. Values presented in parenthesis are an acceptable range forthe given element, unless stated otherwise.

EXAMPLE 1 Primary and Secondary Culture of Normal Human EpidermalKeratinocytes in HECK-109 Serum-Free Medium

Isolation of Basal Cells and Primary Cultures

Primary cultures of normal human basal epidermal keratinocytes werestarted by subjecting full-thickness skin samples to enzymaticdigestion. Skin obtained from biopsies or autopsies was first cleaned ofadhering subdermal fat and the dermis was reduced to less than 3 mm inthickness. The skin sample was then cut into 8 to 12 small pieces(usually 0.5 cm²). These pieces were floated on top of sterile CCS (CellCompetency Solution). CCS consisted of glucose, 10 mM; KCl, 3 mM; NaCl,130 mM; Na₂ HPO₄, 7H₂ O, 1 mM; phenol red, 3.3 μM; HEPES at 23 mM; (SeeShipley, G. D. and Ham, R. G., In Vitro 17:656-670 (1981)) and 0.17%trypsin (w/v) and 100 units/ml of both penicillin and streptomycin.After 14 to 16 hours of digestion at 4° C., the dermis was separatedfrom the epidermis by a split-dermis technique. This was accomplished byplacing the cornified side of the epidermis on a clean sterilepolystyrene surface whereupon the epidermis spontaneously detaches, andthe dermis is removed with sterile forceps. Trypsin digestion cleavesthe skin along a fracture line which separates some of the basal cellswith the dermis, but frees other basal cells lying between the dermisand the fracture line just above the basal cell layer.

The trypsin-treated epidermis, so split from the dermis, was enrichedfor a subpopulation of loosely-associated, clonally competent basalcells. In a series of experiments, the inventor herein discovered thatthese loosely-associated basal cells are larger than the basal cellsthat remain associated with the dermis. Moreover, these larger basalcells are separable by cell sorting procedures using afluorescence-activated cell sorting device. They also have a greatercolony-forming ability than the dermis-associated basal cells, asdemonstrated by clonal growth experimentation.

The loosely-associated basal cells were collected in ice-cold (0°-4° C.)CCS containing 0.1-1.0% w/v SOTI solution in place of the trypsin. Thecell suspension was then filtered on ice through a 100 micrometer sizedNylon mesh using sterile procedures. Filtration removes the cellaggregates and ensures preparation of a single cell suspension. Thecells were pelleted by low speed centrifugation (800×g (gravity), 5mins.) at 4° C. The CCS containing SOTI was aspirated off and theremaining cells were resuspended by gentle pipetting in CCS, and washedonce with ice-cold HECK-109 (serum-free basal nutrient medium; seeExample 2 for detailed preparation of this medium). The centrifugationstep was repeated as above, and the resulting cell pellet wasresuspended in 1 to 2 ml of HECK-109. Cell counts were obtained bystandard cell chamber counting methods. Primary cultures were initiatedinto HECK-109 FS supplemented with 0.1 (0.05-0.20) mM ethanolamine; 0.1(0.5-0.20) mM phosphoethanolamine; 0.5 (0.1-1.0)μM hydrocortisone; and 5ng/ml EGF. Antibiotics which were added at this time can be removed 2 to3 days later when the proliferating cell cultures are refed freshHECK-109 FS. The two protein growth factors (EGF and IGF-1) were addedaseptically to the medium. All media was sterilized through acommercially available membrane filter (0.2 microns). The initialseeding density for initiating the primary culture is 5×10³ basal cellsper cm² tissue culture flask. Two flasks were set up from an initialyield of 1 to 2×10⁶ cells isolated from the 2 cm² piece of skin. Itshould be appreciated that the same isolation procedure used for basalkeratinocytes from skin can be used to obtain other basal epitheliacells from tissues such as cornea, gingiva, ureter and the like.

Secondary Culture Procedure--Secondary cultures may be initiated fromeither primary cultures or early passage secondary cultures. Earlypassage secondary cultures were passaged by enzymatic dissociation ofcells. This serial passage technique is not standard. It involves theuse of ice-cold 0.02% (0.02-0.20) SOTI (w/v) in CCS as detailed abovefor initiating primary cultures. Secondary cultures were seeded at aninitial cell density of 1000 cells per cm². Lower seeding densities arealso acceptable.

The procedure for calculating colony forming efficiency (CFE) of thebasal cells recovered from the epidermis and used to initiate a primaryculture is based upon setting up duplicate primary cultures at 5000cells per cm² as described above, and then to count the number of cellswhich attach and later form a colony of at least 8 or more cells, threedays after seeding the primary culture. By this method, the percentattachment of epidermal cells was 50 to 60 percent of the input cells.

EXAMPLE 2 Preparation of HECK-109 Basal Nutrient Medium

One aspect of the present invention relates to the preparation of a newmedia suitable for the large scale amplification of both primary andsecondary cultures of normal human epithelial cells, such askeratinocytes, and for conversion of proliferating normal humanepithelial monolayer cultures to a fully differentiated tissuetransplantable to a human being. More particularly, this Example 2 isdirected to the materials and procedures for preparation of a basalnutrient medium (Human Epidermal Cell Keratinocyte, HECK-109), andexperiments evidencing its superiority in stimulating epithelial cellgrowth. The media according to this invention are novel and unobvious bydesign of the osmolarity, toxicity and pH-buffering properties.

Table I below details the concentration of components in basal medium,HECK-109. All biochemicals, growth factors and hormones were purchasedfrom Sigma Chemical Company (St. Louis, Mo., U.S.A.), and all inorganicchemicals were from Fisher Scientific (Pittsburgh, Pa., U.S.A.). Alltrace elements in Stock T were from Aesor (Johnson Matthey, Inc.,Seabrook, N.H., U.S.A., Purotronic Grade). EGF was prepared according tothe procedure of Savage, R. C. and Cohen, S. (J. Biol. Chem.247:7609-7611 (1972)), or purchased from Collaborative Research, Inc.,Waltham, Mass.

One liter of HECK-109 was prepared in a separate stock solution fashionas described in Table 1 with respect to Stocks 2 through 10. MediumHECK-109 differs from all other media in the part art by its Stock 1amino acids, its concentration of NaCl (113 mM; Range 90-140) and HEPES(20 mM; Range 14-22). The concentration of the six (6) amino acids iscritical and must be within the following ranges: isoleucine at0.5-5.0×10⁻⁴ M; histidine at 0.5-2.5×10⁻⁴ M; methionine at 1.0-5.0×10⁻⁴M; phenylalanine at 1.0-5.0×10⁻⁴ M; tryptophan at 0.5-5.0×10⁻⁴ M;tyrosine at 1.0-5.0×10⁻⁴ M.

                  TABLE 1                                                         ______________________________________                                        Composition of Basal Nutrient Medium HECK-109                                                         Concentration                                                                 in Final Medium                                       Stock  Component        mg/l      mol/l*                                      ______________________________________                                        1      Arginine.HCl     421.4     2.00 × 10.sup.-3                             Histidine.HCl.H.sub.2 O                                                                        36.1      1.70 × 10.sup.-4                             Isoleucine allo-free                                                                           33.0      1.50 × 10.sup.-4                             Leucine          132.0     1.00 × 10.sup.-3                             Lysine.HCl       36.6      2.00 × 10.sup.-4                             Methionine       45.0       3.0 × 10.sup.-4                             Phenylalanine    50.0       3.0 × 10.sup.-4                             Threonine        23.8      2.00 × 10.sup.-4                             Tryptophan       40.8      2.00 × 10.sup.-4                             Tyrosine         54.0       3.0 × 10.sup.-4                             Valine           70.2      6.00 × 10.sup.-4                             Choline Chloride 27.9      2.00 × 10.sup.-4                             Serine           126.1     1.20 × 10.sup.-3                      2      Biotin           0.0146    6.00 × 10.sup.-8                             Calcium Pantothenate                                                                           0.285     1.00 × 10.sup.-6                             Niacinamide      0.03363   3.00 × 10.sup.-7                             Pyridoxal.HCl    0.06171   3.00 × 10.sup.-7                             Thiamine.HCl     0.3373    1.00 × 10.sup.-6                             Potassium Chloride                                                                             111.83    1.50 × 10.sup.-3                      3      Folic Acid       0.79      1.80 × 10.sup.-6                             Na.sub.2 HPO.sub.4.7H.sub.2 O                                                                  536.2     2.00 × 10.sup.-3                      4a     Calcium chloride.2H.sub.2 O                                                                    14.7      1.00 × 10.sup.-4                      4b     Magnesium chloride.6H.sub.2 O                                                                  122.0     6.00 × 10.sup.-4                      4c     Ferrous sulfate.7H.sub.2 O                                                                     1.39      5.00 × 10.sup.-6                      5      Phenol red       1.242     3.30 × 10.sup.-6                      6a     Glutamine        877.2     6.00 × 10.sup.-3                      6b     Sodium pyruvate  55.0      5.00 × 10.sup.-4                      6c     Riboflavin       0.03764   1.00 × 10.sup.-7                      7      Cysteine.HCl     42.04     2.40 × 10.sup.-4                      8      Asparagine       13.2      1.00 × 10.sup.-4                             Proline          34.53      3.0 × 10.sup.-4                             Putrescine       0.1611    1.00 × 10.sup.-6                             Vitamin B.sub.12 0.407     3.00 × 10.sup.-7                      9      Alanine          8.91      1.00 × 10.sup.-4                             Aspartic Acid    3.99      3.00 × 10.sup.-5                             Glutamic Acid    14.71     1.00 × 10.sup.-4                             Glycine          7.51      1.00 × 10.sup.-4                      10     Adenine          12.16     9.00 × 10.sup.-5                             Inositol         18.02     1.00 × 10.sup.-4                             Lipoic Acid      0.2063    1.00 × 10.sup.-6                             Thymidine        0.7266    2.00 × 10.sup.-6                      Trace  Copper sulfate   0.0025    1.00 × 10.sup.-8                      Element                                                                              Selenic Acid     0.00687   3.00 × 10.sup.-8                             Manganese Sulfate.5H.sub.2 O                                                                   0.000241  1.00 × 10.sup.-9                             Sodium Silicate.9H.sub.2 O                                                                     0.001421  1.00 × 10.sup.-7                             Ammonium Molybdate.4H.sub.2 O                                                                  0.00124   1.00 × 10.sup.-9                             Ammonium Vanadate                                                                              0.00059   1.00 × 10.sup.-9                             Nickel Chloride.6H.sub.2 O                                                                     0.00012   5.00 × 10.sup.-9                             Stannous Chloride                                                                              0.000113   5.00 × 10.sup.-10                           Zinc Chloride.7H.sub.2 O                                                                       0.1438    5.00 × 10.sup.-7                      Solids S                                                                             Glucose          1081.0    6.00 × 10.sup.-3                             Sodium Acetate.3H.sub.2 O                                                                      500.0     3.70 × 10.sup.-3                             Sodium Bicarbonate                                                                             1176.0    1.40 × 10.sup.-2                             Sodium Chloride  6600.0    1.13 × 10.sup.-2                             HEPES            4700.0    2.00 × 10.sup.-2                      ______________________________________                                         *All above components come together to a final volume of 1 liter of           distilled and 0.22 μmfiltered water.                                  

The concentrations of these six (6) important amino acids have beenshown by the inventor to be necessary for sustained basal cellproliferation. By further experimentation, the inventor discovered thatsuperior growth occurs when the osmolarity of the media are between 275and 310 milliosmoles (mosmols). The osmolarity of the inventive mediaare critical to proper cell growth.

Through an extensive series of clonal growth experiments in which theosmolarity was held constant at 300 mosmols and the concentration ofHEPES varied between 14 to 28 mM, it was also discovered that theinventive media must incorporate HEPES at between 14-22 mM, preferablybetween 18 and 22 mM with 22 mM being the most preferred. This is alsocritical to the media of this invention. Table 2 presents results ofclonal growth experiments showing that the design of HECK-109 supports ahigher growth rate and a higher colony forming efficiency than astandard MCDB 153 commercial medium.

A most significant aspect of the present invention is that theconcentration of 14 to 22 mM concentration HEPES in HECK-109 mediumresults in a 2 to 3 fold higher colony forming efficiency than thatpreviously attainable. The second significant discovery is that anosmolarity of 280-310 mosmols, most preferably 300 mosmols, of the mediapermits attainment of higher saturation densities at confluence of themonolayer culture. The third significant discovery is that it isnecessary to provide the indicated concentrations of 6 key amino acidspresent in Stock 1 (typically 2 to 5 times higher concentration thanthat in commercially available in MCDB 153 medium). This allows humanepithelial cell cultures to routinely achieve a cell density equal to orgreater than 100,000 cells per cm². HECK-109 incorporates these threediscoveries in such a way that the media will allow for and fullysupport the formation of a complete reformed human epithelium asdetailed below.

                  TABLE 2                                                         ______________________________________                                        Effect of Osmolarity and HEPES Concentrations on the Growth                   Response of Normal Human Keratinocytes                                                                        Growth Response/                              Culture HEPES    NaCl   Osmolarity                                                                            (Colonies                                                                            dish)                                  Media   (mM)     (mM)   (mosmols)                                                                             AKH.sup.a                                                                            NHK.sup.b                              ______________________________________                                        MCDB-153                                                                              28       130    340      84 ± 12                                                                          275 ± 24                            HECK-109                                                                              22       104    300     196 ± 23                                                                          438 ± 35                            ______________________________________                                         .sup.a Secondary cultures of adult skin normal human keratinocytes (AHK)      were seeded at 2 × 10.sup.3 cells/dish in MCDB 153 medium and refed     HECK109 48 hours later. Dishes were fixed for colony counts 6 days later.     .sup.b Clonal growth experiments were performed on neonatal foreskin          secondary normal human keratinocytes (NHK) cultures as described in Wille     J.J. et al., J. Cellular Physio. 121:31-44 (1984).                       

An additional experiment was conducted to investigate the effect ofvarying HEPES levels on the clonal growth response of normal humankeratinocytes cultured on complete MCDB 153 medium prepared with 104 mMNaCl and containing five (5)different HEPES concentrations at 300mosmols. Each dish was seeded with 500 cells and allowed to grow for 12days at 37° C. under 5% CO₂.

The cells were fixed for five (5) minutes with ice-cold 2.5%formaldehyde in 1×PBS (phosphate buffered saline), washed twice at roomtemperature with 1×PBS and stained for 15 to 30 minutes with 0.3%crystal violet solution. The dishes were then rinsed under running tapwater to remove excess dye and air dried. Stained colonies were countedwith an ARTEX bacterial colony counter (Model 870). The background noiseof approximately ten (10) was corrected and instrument size and levelsettings set at 0.27 and 0, respectively.

    ______________________________________                                        HEPES Concentration                                                                          Clonal Growth Response                                         (mM)           (colonies/dish)                                                ______________________________________                                        28              187 ± 6.4                                                  25              179 ± 2.0                                                  23             211 ± 11                                                    20             243 ± 26                                                    14             295 ± 16                                                    ______________________________________                                    

These results indicate that as the HEPES concentration decreases, theclonal growth increases wherein the relationship of HEPES concentrationto clonal growth can be expressed as y=-8.56x+111.4.

An additional experiment compared the osmolarity of MCDB 153 at 340mosmols and HECK-109 at 300 mosmols on the cell densities of normalhuman keratinocytes after three (3) days of growth. Cells were seededinto 25 cm² flasks at a cell density of 1×10³ cells/cm² for four (4)days earlier and fed MCDB 153, then on day 0 (cell density of 6.7cells/cm² ×10³), cultures were fed either HECK-109 or MCDB-153. On day3, the cell density of the HECK-109 FS (300 mosmols) was 44×10³ cellsper cm² while the MCDB-153 (340 mosmols) was 37.2×10³ cells/cm². Thisdemonstrates the importance of the correct osmolarity for the propergrowth of the cells.

EXAMPLE 3 Clonal Growth Studies employing Single Cell Clones in HECK-109Medium

Human keratinocyte cultures were initiated, from either foreskin oradult female breast skin, as detailed above in Example 1, and thenplaced into secondary culture in complete HECK-109 FS medium. Thepurpose of the following experiment was to determine the colony formingability of individual keratinocyte stem cells obtained from differentskin donors and from different passage levels of the same normal humankeratinocyte sample. It is stressed here that each culture wasestablished from a single genetic source to ensure that the responsesobserved represent only deliberate experimental manipulations. Thetechnique of cloning individual cells was accomplished by seeding 1000cells from a exponentially dividing parent culture into a 100 mm² Petridish containing prewarmed HECK-109 FS medium. Visual observations ofeach such single cell isolate were made and a daily record of the numberof cells formed from each single-celled clone. The results of theseexperiments are set forth in Tables 3 and 4. The data indicate that eachproliferating basal cell from a given donor culture has an exceedinglyhigh clonogenic potential.

Typically, a clone is comprised of more than 1000 cells, indicating thatthe original single cell had undergone more than 10 doublings. Suchclones are, by definition, basal stem cells and data on their clonalanalysis is presented in Tables 3 and 4. The results in Table 3 showthat 70% of single cells derived from a third passage neonatal foreskinnormal human keratinocyte cultures were, in fact, keratinocyte stemcells. Adult-derived normal human keratinocyte secondary cultures alsoat the third passage level had a significantly reduced clonogenicpotential (48%), which correlates with the slower growth rate (48 hourdoubling time) of the parent culture, which, when compared with therapid (24 hour doubling time) of the neonatal foreskin normal humankeratinocyte culture, clearly shows that the proliferative potential ofstem cells is determined by prior culture conditions. Table 4 presentsdata comparing five (5) different neonatal foreskin normal humankeratinocyte cultures and shows again, the fact that a consistently highclonogenic potential is maintained in secondary cultures under priorculture conditions.

                  TABLE 3                                                         ______________________________________                                        Comparison of the Proliferative Potential of Individual Adult Versus          Neonatal Keratinocyte Basal Cells                                             Prior Culture Condition.sup.ab                                                                  Cell Density                                                                            Average GT                                                                            % Proliferative                           Clone No.                                                                             Passage No.                                                                             (10.sup.4 /cm.sup.2)                                                                    (hrs)   Clones (N)                                ______________________________________                                        Adult   3         0.4       48      48 (109)                                  Neonatal                                                                              3         7.5       24      70 (106)                                  ______________________________________                                         .sup.a GT is defined as the average population doubling time (in hours) o     the culture.                                                                  .sup.b N is the number of single cell clones tested.                     

                  TABLE 4                                                         ______________________________________                                        Clonal Analysis of the Proliferative Potential of Individual                  Keratinocyte                                                                  Basal Cells                                                                   Prior Culture Condition.sup.a b                                               Neonatal                                                                      clone   Passage Cell density        % Proliferative                           No.     level   (10.sup.4 /cm.sup.2)                                                                    Average (GT(hr)                                                                         clones (N)                                ______________________________________                                        1       2       1.87       24 (5).sup.c                                                                           75 (32)                                   1       3       1.73      24 (6)    66 (35)                                   2       2       1.0       24 (4)    79 (34)                                   3       2       1.1       24 (6)    68 (37)                                   4       2       0.65      30 (4)    51 (93)                                   ______________________________________                                         Mean % = 63 (231)                                                             .sup.a GT is defined as the average population doubling time (in hours) o     the culture.                                                                  .sup.b N is the number of single cell colonies tested.                        .sup.c The number in parenthesis within this column indicates the age of      the parent culture in days.                                              

In summary, the combined results of 231 single cells cloned at randomfrom secondary cultures reared in HECK-109 FS medium showed that atleast 63% were keratinocyte stem cells. The results of these single cellclonal studies indicate that the novel basal medium HECK-109 supportsincreased clonal growth of basal cells and enhances their clonogenicpotential 10 times above the reported values obtained by Green et al. inU.S. Pat. No. 4,016,036, 1980 or in the serum-free culturing process ofBoyce et al., U.S. Pat. No. 4,673,649. These considerations are ofutmost relevance to the present invention and a major advancement in thestate of the art. Further, the present invention allows commerciallyusable in vitro manufactured living skin substitutes, cornealsubstitutes, gingival substrates and other epithelium.

EXAMPLE 4 Formation of a Complete Epidermis in the Serum-Free HECK-109Culture Medium

The formation of a complete reformed human epidermis in serum-freeHECK-109 medium was accomplished in three separate culture phases. PhaseI of the culture began with the seeding of basal keratinocyte stem cellsisolated using CCS into culture dishes at a cell density ofapproximately 1000 cells per cm as set forth in Example 1. Typically,several million keratinocyte stem cells were prepared from a singleprimary culture flask, representing about a 5000-fold increase in cellsover the starting stem cells recovered from the skin sample. All normalhuman keratinocyte cultures were fed HECK-109 FS medium as set forth inTable I. Cultures were refed HECK-109 FS every other day until the celldensity was 1 to 2×10⁴ cells per cm². Cultures fed HECK-109 FS everyother day typically reach confluence in six (6) to ten (10) days.

Phase II, the induction of the stratum spinosum and the stratumgranulosum and concomitant maintenance of the stratum germinativum,begins with the removal of the HECK-109 FS and its replacement withHECK-109 DM. The use of HECK-109 DM overcomes the completeparasynchronous growth arrest in the G₁ phase of the cell cycle as seenby Shipley, G. D., et al., Cancer Res. 46:2068-2071 (1986) and Wilke, M.et al., Amer. J Pathol 131:171-181 (1988). The addition of HECK-109 DMto confluent monolayer cultures, induced within 48-96 hours aprogressive stratification of the basal cells to form a multilayeredepithelium. Concomitantly, the clonogenic potential of the culturedeclined only to about 50%. HECK-109 DM allows a fraction of the basalcells to escape growth arrest while the remaining basal cells go on toform the non-dividing suprabasal layer. The suprabasal cellsprogressively enlarge into differentiated cell types representative ofthe spinous and granular cell layers, and migrated to the upper layersof the multilayered epidermis where they were shed into the medium. Theresult of this differentiation process was the formation of an extendedsheet of multilayered epidermis (end of Phase II cell culture). Thisprocess takes about a week to complete and resulted in an incompleteliving epidermis comprised of a basal cell layer with an overlyingMalphigian cell layer (stratum germinativum+stratum spinosum).

The final step of the culture process (Phase III) converted theincomplete epidermis to a complete human epidermis by induction in theuppermost layers of a cornified cell layer (stratum lucidum, stratumcorneum and stratum disjunction). This step was accomplished by removalof the HECK-109 DM and its replacement with HECK-109 CM. During PhaseIII of culture, granular cells continued to mature into cornified,anucleate cells which formed the topmost layer of the completedepidermis.

EXAMPLE 5 Applications of Reformed Human Epidermis as a Living SkinSubstitute

From previous studies in the literature, it is widely known that humanskin is a target organ for certain sex steroid hormones. In fact, skinis the next most active site after the liver for the metabolicinterconversions of steroid hormones. Nevertheless, little is knownabout the direct effect of sex steroid hormones such as testosterone,progesterone and estrogens on the growth and differentiation of normalhuman keratinocytes.

A. EFFECT OF SEX STEROID HORMONES ON BASAL EPIDERMAL CELLS CULTURES INSERUM-FREE MEDIUM.

It has been reported (Peehl, D. M. and Ham, R. G. In Vitro 16:516-525(1980)) that 17- β-estradiol stimulated the growth of epidermal cells inculture. However, the stimulatory effect that was observed was minimaland occurred under less than optimal clonal growth conditions. It isbelieved that the medium employed by Peehl et al. and the growth factorspresent in that medium limited the effectiveness of the 17-β-estradiol.In view of these considerations and because living skin substitutes arean ideal model for assaying the effects of sex steroid hormones, it wasdecided to measure the effects of sex steroid hormones in HECK-109. Thisexperiment was conducted to investigate the effect of testosterone,progesterone, 17-β-estradiol on the clonal growth of normal humankeratinocytes in HECK-109 FS. The data contained in Table 5 evidencethat both estradiol (3.4×10⁻⁶ M) and progesterone (3.7×10⁻⁶ M) exert aninhibitory action on the proliferation of basal keratinocyte stem cellsderived from either newborn foreskin or adult breast skin. In contrast,testosterone (3.7×10⁻⁶ M) had only a negligible effect on the clonalgrowth of these cells. Further, the results show that female-derivedkeratinocytes are less sensitive to the inhibitory effect of the femalesex steroid hormones than are the male-derived keratinocytes (providedthat the keratinocytes derived from adult skin are also for some unknownreason less sensitive than newborn). The keratinocyte proliferation maybe profoundly perturbed by continuous exposure to progesterone orprogesterone-related steroids and therefore, these effects may need tobe taken into account where reformed human epidermis is used as a modelfor the transdermal delivery of contraceptive steroids.

                  TABLE 5                                                         ______________________________________                                        Effect of Estradiol, Progesterone, and Testosterone on Clonal Growth of       Norman Human Basal Keratinocytes                                                                  Growth Responses.sup.a                                                        (colonies/dish)                                           Culture Conditions  AH.sup.b                                                                             NF.sup.c                                           ______________________________________                                        HECK-109 FS medium  569    286                                                                    585    312                                                +Testosterone       603    259                                                (1.0 μg/ml)      583    264                                                +Progesterone       311    58                                                 (1.0 μg/ml)      402    26                                                 +Estradiol          426    83                                                 (1.0 μg/ml)      363    58                                                 ______________________________________                                         .sup.a Values represent the results of duplicate determinations.              .sup.b AH, adult skin keratinocytes were seeded at a density of 1000 cell     per dish; the dishes were fixed and counted 10 days later.                    .sup.c NF, foreskin keratinocytes were seeded at a density of 500 cells       per dish; the dishes were fixed and counted 10 days later.               

EXAMPLE 6 Demonstration of Specific and Saturable 17-β-EstradiolReceptors in Pre-Formed Human Epidermis

Human epidermis prepared in accordance with the present invention wasused as a tool to assay the affect of a wide variety of test substances,e.g., hormones, toxins, viruses and carcinogens. Of immediate interestis whether reformed human epidermis has specific and saturable sexsteroid hormone binding sites.

A series of experiments were conducted to measure the binding ofradiolabelled 17-β-estradiol to replicate samples of human epidermisfrom a single genetic source. Reformed human epidermis was produced byculturing basal keratinocytes as outlined in Example 4 in replicate24-well cluster dishes (Corning Tissue Culture Wares, Corning, N.Y.,U.S.A.) through Phase III of the culture. Several test wells weresampled at the time of the binding experiments by standard histologicalmethods to verify that a complete epidermis had, indeed, been produced.The conditions of the binding assay were as follows: Phase III culturemedium was aseptically removed and to the reformed human epidermis ineach well, 0.5 ml of CCS containing 10 to 50 nmol of radiolabelled 17β-estradiol (160 Ci/mM;0.2 μCi/ml) was added. The radiolabelledestradiol was purchased from New England Nuclear Corporation, Boston,Mass., U.S.A. The concentration of radiolabelled estradiol was fixed athalf maximal saturation to assure effective competition with unlabelledidentical and analogue steroid hormones over a wide range of competitorconcentrations. The sex steroid competitors tested in the competitionbinding assay were 17-β-estradiol and other analogues such astestosterone, estriol, levonorgestral and norethisterone. At the end ofthe 20 hour incubation interval (at 4° C.), the radiolabelled solutionswere removed, the surface of the reformed human epidermis samples wererinsed gently with 1 ml of ice-cold CCS and 0.5 ml of Type IVcollagenase (Dispase, 20 U/ml, Boehringer-Manheim, Los Angeles, Calif.,U.S.A.) was added to each well to enzymatically release the intactepidermal sheet. The released reformed human epidermis from eachtreatment well was transferred to its respective vial and the containedradioactivity was counted in a scintillation spectrometer.

Only 17-β-estradiol was an efficient competitor for the 17-β-estradiolreceptor. Estriol a close structural analog of estradiol also showedsignificant competition while testosterone and the progesteroneanalogues were not competitive. These results demonstrate that reformedhuman epidermis produced as intact epidermal sheets according to thepresent invention is a good model for biochemical assay of steroid sexhormone receptors. The results of this experiment also provide directevidence for the functional fidelity of reformed human epidermis as aliving skin substitute.

EXAMPLE 7

This experiment was conducted to compare the serum-free basal nutrientHECK-109 to a serum-free, commercially available basal nutrient mediaknown as MCDB 153. The inventive media, HECK-109, was compared side byside with the same cells under identical conditions and mediasupplements. Basal nutrient HECK-109, was prepared according to Example2 and its osmolarity was found to be 304 mosmols. The basal media MCDB153 was prepared and had an osmolarity of 346 mosmols.

Normal human foreskin keratinocytes were prepared as described inExample 1 and in conformance with an article published by Wille et al.,Journal of Cellular Physiology, 121:31-44 (1984). Primary cultures wereharvested at about 50% confluence and were serial passaged into eitherserum-free MCDB 153 media or HECK-109 media. 100 cells were seeded intoplastic, disposable Petri dishes containing media supplemented withethanolamine at 0.1 mM; phosphoethanolamine at 0.1 mM; hydrocortisone at0.1M; and 0.1 mM calcium chloride. The media also contained 5 μg/mlinsulin. Duplicate dishes were then further supplemented with either noepidermal growth factor (EGF) or 0.01, 0.1, 1.0 or 10 ng/ml of EGF. Thedishes were incubated for 10 days at 37° C. with 5% CO₂ in a humidifiedincubator. The cells were then fixed and stained for macroscopicobservation of colonies.

In every Petri dish that was fed HECK-109 medium, there was betterclonal growth (more and larger colonies) as compared to the dishes fedMCDB 153 media. In addition, even in the media that did not contain EGFsupplementation, HECK-109 colonies evidenced more and larger coloniesand therefore demonstrated better clonal growth. There was an increasinggrowth response with increasing levels of EGF concentration up to 1ng/ml EGF.

EXAMPLE 8

The experiment of Example 7 was repeated except that the HECK-109 mediawas prepared with 1× and 2× amounts of Stock 1 of the amino acids setforth in Table 1. The results of this experiment evidenced that HECK-109supplemented with 1× and 2× of the Stock 1 amino acids resulted inbetter clonal growth than the commercially available MCDB 153 media.

EXAMPLE 9 Autologous Cultivated Corneal Epithelium

This experiment investigates the restoration of the human cornealsurface with autologous corneal epithelial sheets generated by serialcultivation of limbal cells. Cells are cultivated from a 1 mm squarebiopsy sample taken from the limbus of the healthy eye of a patient withsevere alkali burns. The procedure set forth in Example 1 for theisolation of the basal cells is conducted using the CCS. The formationof a complete reformed human cornea in serum-free HECK-109 media, wasaccomplished in three (3) separate culture phases. The process set forthin Example 4 was utilized except that the final step (Phase III) was notutilized to create a cornified cell layer. Graphs are prepared fromconfluent secondary cultures which are released from the plastic dishwith the neutral protease Dispase II (Boehringer-Manheim, Germany) andmounted on a petrolatum gauze about 1.5 by 1.5 cm. The graphs typicallycontain about 2×10⁶ cells and are about 1 cm². The graphs are thenapplied to the surgically ablated area of the eye.

The injured eye is prepared for transplant, draped in topical anesthesiawith a lid block by 2% plain xylocaine. The conjunctival epitheliumcovering the cornea and limbus is removed with a blunt knife andscissors and a 360° peritomy extending for at least 2 mm beyond thelimbus is conducted. The cultured epithelial graph is placed on theprepared eye, mounted on a petrolatum gauze, which is gently removedunder a microscope immediately after grafting. A soft, therapeutichydrophilic contact lens is then placed over the graft.

After grafting, the eye is patched tightly for three (3) days. Thetherapeutic contact lens is removed two (2) weeks after grafting.

The autologous cultured corneal epithelium prepared in accordance withthis invention can restore the corneal surface of the patient withcomplete loss of the corneal-limbus epithelium. Long term follow up willshow the stability of regenerated corneal epithelium and a strikingimprovement in the patient's comfort and visual acuity.

From the experiment, it will be shown that enough epithelium to coverthe entire corneal-limbal epithelial surface can be obtained from a 1 mmsquare limbal biopsy sample, allowing minimal stem cell depletion fromthe healthy eye. This investigation will also demonstrate thatautologous grafts prepared from limbal cultures in accordance with themethods and media of this invention, will generate an authentic cornealepithelium for patients.

EXAMPLE 10 Normal Human Gingival Keratinocyte Production

Gingivectomies are commonly performed on humans suffering fromgingivitis. The excision of a portion of the gingiva for periodontaldisease such as the inflammation of the gums, often leaves the patientwith severely impaired oral hygiene. Through the use of the media andprocess of the present invention, patients prior to undergoing such adental procedure, may have prepared in vitro, autologous tissue that canbe used to replace the diseased tissue. Thus, in accordance with thisinvention, serum-free cultures of normal human gingival keratinocytesare prepared in a manner that begins with the isolation of competentbasal cells through the use of a technique described in Example 1. Theobtained basal cells are then serially passaged into the serum-freeHECK-109 media as set forth in Example 4. The passaged cells are seededonto plastic tissue culture dishes at 5×10⁴ cells per cm² and fed freshHECK-109 FS medium every other day. At the conclusion of the thirdphase, the human gingival keratinocytes will be studied by light andelectron microscopy and will possess typical epithelial features such asdesmosomes and perinucleartonofilaments The prepared tissue will reactpositively to monoclonal antibodies specific for basal layer cells ofthe human epidermis. Thus, through the process and media of the presentinvention, a parakeratotic stratified epithelial sheet can be producedin vitro by suitable culture manipulations which then will be useful fortransplantation therapy or the tissues will provide a useful model forbacterial epithelial interactions and the effects of toxic substances onoral epithelium.

EXAMPLE 11 Growth of Ureteral Epithelial Cells in Serum-Free Culture

The term "urinary tract infection" describes a heterogeneous group ofdisorders localized to the urinary tract. Gram negative bacteria are thedominant cause of urinary tract infections. The initial pathogenic eventin the urinary tract occurs on the mucosal surface and is the attachmentof bacterial to the host mucosa. Attachment is followed by mucosalinflammation and shedding viable uroepithelial cells. Continuedinfection often results in the destruction of the ureter and a methodfor the autologous production of replacement tissue would be highlydesirable.

Thus, in this experiment, cells are isolated from human ureters that areobtained after surgical removal. The basal cells from the normal humanuroepithelium are isolated as set forth in Example 1. The formation of acomplete uroepithelium is conducted in a manner similar to that setforth in Example 4. The sheet of human uroepithelium can then be used totest the effects of various pathogens on this tissue or can be used as atransplant material for damaged tissue.

Industrial Applicability

The present invention is directed to the design and formulation of thevarious novel HECK 109 media which provide for the differentiation ofpluripotent basal epithelial cells to a fully differentiated humanepithelium in vitro. HECK-109 is the basal medium for cell starting:HECK-109 FS is for control of cellular growth; HECK-109 DM is for theinduction of differentiation and formation of a Malphigian layer andHECK-109 CM is designed for the induction of cellular differentiation ina pre-existing reformed tissue produced by HECK-109 DM. The inventionalso relates to a method of sequential control for the in vitroconstruction of a histologically complete living epithelium. The tissuederived from the media and methods of the invention have application inin vitro testing of pharmaceuticals and topical drugs; screening oftoxicants, carcinogens, complete or incomplete tumor promoters;evaluation of infective human agents including viruses, e.g., humanpapilloma viruses, Herpes-simplex viruses and Epstein-Barr virus; andscreening of cosmetics.

Most importantly the present invention allows for the use ofautologously-derived tissue for transplantation in the treatment ofburns or other trauma. Further, the present invention would allow forautologous production of corneal tissue, gingival tissue, ureter tissueand other epithelium for transplant to a patient in need thereof.

Numerous modifications and variations in the invention are expected tooccur to those skilled in the art upon considerations of the foregoingdescriptions. The invention should not be construed as limited to thepreferred embodiments and modes of preparation described herein, sincethese are to be regarded as illustrative rather than restrictive.

We claim:
 1. An aqueous solution for isolating epithelial cells fromanimal tissue, said solution comprising:a) glucose at a concentration ofabout 10 mM; b) N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) ata concentration of 16-22 mM; c) sodium chloride at a concentration of90-140 mM; d) potassium chloride at a concentration of about 3 mM; e)sodium orthophosphate (Na₂ HPO₄.7H₂ O) at a concentration of 1 mM; f)phenol red at a concentration of 0.0033 mM; g) about 100 units ofpenicillin per ml of solution; h) about 100 units of streptomycin per mlof solution; and i) one component selected from the group consistingof:(i) trypsin at a concentration of 0.1%-0.2% w/v; and (ii) soy beantrypsin inhibitor at a concentration of 0.1-1.0% w/v.
 2. The solution ofclaim 1 wherein said component is soybean trypsin inhibitor.
 3. Thesolution of claim 1 wherein said component is trypsin.
 4. The solutionof claim 1 wherein:a) said sodium chloride is at a concentration of 100to 130 mM; b) said N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid)(HEPES) is at a concentration of 18 to 21 mM; c) said trypsin is at aconcentration of 0.12 to 0.18% w/v; and d) said soybean trypsininhibitor is at a concentration of 0.3 to 0.8% w/v.
 5. A method for theisolation of basal epithelial cells from animal tissues, said methodcomprising the steps of:a) obtaining animal epithelium; b) comminutingsaid epithelium; c) placing said comminuted epithelium solutiondescribed in an aqueous solution according to claim 3 at a temperatureand for a time sufficient to allow separation of the basal epithelialcells from the epithelium; d) collecting said epithelial cells; and e)passaging said basal epithelial cells to a solution containing soybeantrypsin inhibitor.
 6. A serum-free medium for culturing animalepithelial cells comprising:a)N-(2-OH-ethyl-)piperazine-N'-(2-ethane-sulfonic acid) at a concentrationof 14-22 mM; b) sodium chloride at a concentration of 100-120 mM; c)histidine at a concentration of 0.1-0.25 mM; d) isoleucine at aconcentration of 0.05-0.5 mM; e) methionine at a concentration of0.1-0.5 mM; f) phenylalanine at a concentration of 0.1-0.5 mM; g)tryptophan at a concentration of 0.05-0.5 mM; and h) tyrosine at aconcentration of 0.1-0.5 mM.
 7. A serum-free medium for culturing animalepithelial cells comprising:a) N-(2-OH-ethyl-)piperazine-N-(2-ethane-sulfonic acid) at a concentration of 14-22 mM; b) sodiumchloride at a concentration of 100-120 mM; c) histidine at aconcentration of 0.1-0.25 mM; d) isoleucine at a concentration of0.05-0.5 mM; e) methionine at a concentration of 0.1-0.5 mM; f)phenylalanine at a concentration of 0.1-0.5 mM; g) tryptophan at aconcentration of 0.05-0.5 mM; h) tyrosine at a concentration of 0.1-0.5mM; and i) insulin like growth factor -1 at a concentration of 0.3-30ng/ml.
 8. A serum-free medium for culturing animal epithelial cellscomprising:a) N-(2-OH-ethyl-)piperazine-N'-(2-ethane-sulfonic acid) at aconcentration of 14-22 mM; b) sodium chloride at a concentration of100-120 mM; c) calcium²⁺ ions at a concentration of 0.7-3.0 mM; d)histidine at a concentration of 0.1-0.25 mM; e) isoleucine at aconcentration of 0.05-0.5 mM; f) methionine at a concentration of0.1-0.5 mM; g) phenylalanine at a concentration of 0.1-0.5 mM; h)tryptophan at a concentration of 0.05-0.5 mM; i) tyrosine at aconcentration of 0.1-0.5 mM; and j) beta-transforming growth factor at aconcentration of 3.0-30 ng/ml.
 9. A serum-free medium for culturinganimal epithelial cells comprising:a)N-(2-OH-ethyl-)piperazine-N'-(2-ethane-sulfonic acid) at a concentrationof 14-22 mM; b) sodium chloride at a concentration of 100-120 mM; c)calcium²⁺ ions at a concentration of 0.7-3.0 mM; d) histidine at aconcentration of 0.1-0.25 mM; e) isoleucine at a concentration of0.05-0.5 mM; f) methionine at a concentration of 0.1-0.5 mM; g)phenylalanine at a concentration of 0.1-0.5 mM; h) tryptophan at aconcentration of 0.05-0.5 mM; i) tyrosine at a concentration of 0.1-0.5mM; and j) linoleic acid at a concentration of 1-15 μg/ml.
 10. A methodfor the formation of a histologically complete stratified animalepithelium comprising the steps of:a) isolation of basal stem cells fromanimal epithelium using a solution comprising:i) glucose at aconcentration of about 10 mM; ii)N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at a concentrationof 16-22 mM; iii) sodium chloride at a concentration of 90-140 mM; iv)potassium chloride at a concentration of about 3 mM; v) sodiumorthophosphate (Na₂ HPO₄.7H₂ O) at a concentration of 1 mM; vi) phenolred at a concentration of 0.0033 mM; vii) about 100 units of penicillinper ml of solution; viii) about 100 units of streptomycin per ml ofsolution; and ix) trypsin at a concentration of 0.1%-0.2% w/v; b)recovering said isolated basal stem cells using a solution comprising:i)glucose at a concentration of about 10 mM; ii)N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at a concentrationof 16-22 mM; iii) sodium chloride at a concentration of 90-140 mM; iv)potassium chloride at a concentration of about 3 mM; v) sodiumorthophosphate (Na₂ HPO₄.7H₂ O) at a concentration of 1 mM; vi) phenolred at a concentration of 0.0033 mM; vii) about 100 units of penicillinper ml of solution; viii) about 100 units of streptomycin per ml ofsolution; and ix) soy bean trypsin inhibitor at a concentration of0.1%-1.0% w/v; c) culturing said isolated basal stem cells in a mediumto form a confluent sheet of undifferentiated epithelial tissue, saidmedium comprising:i) N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonicacid) at a concentration of 14-22 mM; ii) sodium chloride at aconcentration of 100-120 mM; iii) histidine at a concentration of0.1-0.25 mM; iv) isoleucine at a concentration of 0.05-0.5 mM; v)methionine at a concentration of 0.1-0.5 mM; vi) phenylalanine at aconcentration of 0.1-0.5 mM; vii) tryptophan at a concentration of0.05-0.5 mM; viii) tyrosine at a concentration of 0.1-0.5 mM; and ix)insulin-like growth factor -1 at a concentration of 0.3-30 ng/ml; d)culturing said sheet of undifferentiated epithelial tissue in adifferentiation medium to form a sheet of differentiated and stratifiedtissue, said differentiation medium comprising:i)N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at a concentrationof 14-22 mM; ii) sodium chloride at a concentration of 100-120 mM; iii)calcium²⁺ ions at a concentration of 0.7-3.0 mM; iv) histidine at aconcentration of 0.1-0.25 mM; v) isoleucine at a concentration of 0.1-05mM; vi) methionine at a concentration of 0.1-0.5 mM; vii) phenylalanineat a concentration of 0.1-0.5 mM; viii) tryptophan at a concentration of0.05-0.5 mM; ix) tyrosine at a concentration of 0.1-0.5 mM; and x) betatransforming growth factor at a concentration of 3.0-30 ng/ml; and e)culturing said differentiated and stratified tissue in a cornificationmedium to form a cornified epithelium, said cornification mediumcomprising:i) N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at aconcentration of 14-22 mM; ii) sodium chloride at a concentration of100-120 mM; iii) calcium²⁺ ions at a concentration of 0.7-3.0 mM; iv)histidine at a concentration of 0.1-0.25 mM; v) isoleucine at aconcentration of 0.05-0.5 mM; vi) methionine at a concentration of0.1-0.5 mM; vii) phenylalanine at a concentration of 0.1-0.5 mM; viii)tryptophan at a concentration of 0.05-0.5 mM; ix) tyrosine at aconcentration of 0.1-0.5 mM; and x) linoleic acid at a concentration of1-15 μg/ml.
 11. The method according to claim 10 wherein saidhistologically complete epithelium is human skin.
 12. A method for theformation of a differentiated animal epithelium, said method comprisingthe steps of:a) isolation of basal stem cells from epithelium using asolution comprising:i) glucose at a concentration of about 10 mM; ii)N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at a concentrationof 16-22 mM; iii) sodium chloride at a concentration of 90-140 mM; iv)potassium chloride at a concentration of about 3 mM; v) sodiumorthophosphate (Na₂ HPO₄.7H₂ O) at a concentration of 1 mM; vi) phenolred at a concentration of 0.0033 mM; vii) about 100 units of penicillinper ml of solution; viii) about 100 units of streptomycin per ml ofsolution; ix) trypsin at a concentration of 0.1%-0.2% w/v; b) recoveringsaid isolated basal stem cells using a solution comprising:i) glucose ata concentration of about 10 mM; ii)N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at a concentrationof 16-22 mM; iii) sodium chloride at a concentration of 90-140 mM; iv)potassium chloride at a concentration of about 3 mM; v) sodiumorthophosphate (Na₂ HPO₄.7H₂ O) at a concentration of 1 mM; vi) phenolred at a concentration of 0.0033 mM; vii) about 100 units of penicillinper ml of solution; viii) about 100 units of streptomycin per ml ofsolution; and ix) soy bean trypsin inhibitor at a concentration of0.1%-1.0% w/v; c) culturing said isolated basal stem cells to form aconfluent sheet of undifferentiated epithelial tissue in a mediumcomprising:i) N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at aconcentration of 14-22 mM; ii) sodium chloride at a concentration of90-120 mM; iii) histidine at a concentration of 0.1-0.25 mM; iv)isoleucine at a concentration of 0.05-0.5 mM; v) methionine at aconcentration of 0.1-0.5 mM; vi) phenylalanine at a concentration of0.1-0.5 mM; vii) tryptophan at a concentration of 0.05-0.5 mM; viii)tyrosine at a concentration of 0.1-0.5 mM; ix) insulin-like growthfactor -1 at a concentration of 0.3-30 ng/ml; and d) culturing saidsheet of undifferentiated epithelial tissue to form a sheet ofdifferentiated and stratified tissue in a medium comprising:i)N-(2-OH-ethyl-)piperazine-N'-(2-ethanesulfonic acid) at a concentrationof 14-22 mM; ii) sodium chloride at a concentration of 100-120 mM; iii)calcium²⁺ ions at a concentration of 0.7-3.0 mM; iv) histidine at aconcentration of 0.1-0.25 mM; v) isoleucine at a concentration of0.05-0.5 mM; vi) methionine at a concentration of 0.1-0.5 mM; vii)phenylalanine at a concentration of 0.1-0.5 mM; viii) tryptophan at aconcentration of 0.05-0.5 mM; ix) tyrosine at a concentration of 0.1-0.5mM; and x) beta transforming growth factor at a concentration of 3.0-30ng/ml.
 13. The method according to claim 12 wherein said differentiatedand stratified tissue is selected from cornea and gingiva.